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Oral Polio Vaccine Influences the Immune Response to BCG Vaccination. A NaturalExperiment

Sartono, Erliyani; Lisse, Ida M.; Terveer, Elisabeth M.; van de Sande, Paula J. M.; Whittle,Hilton; Fisker, Ane B.; Roth, Adam; Aaby, Peter; Yazdanbakhsh, Maria; Benn, Christine S.Published in:PLoS ONE

DOI:10.1371/journal.pone.0010328

2010

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Citation for published version (APA):Sartono, E., Lisse, I. M., Terveer, E. M., van de Sande, P. J. M., Whittle, H., Fisker, A. B., ... Benn, C. S. (2010).Oral Polio Vaccine Influences the Immune Response to BCG Vaccination. A Natural Experiment. PLoS ONE,5(5). https://doi.org/10.1371/journal.pone.0010328

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https://doi.org/10.1371/journal.pone.0010328

Oral Polio Vaccine Influences the Immune Response toBCG Vaccination. A Natural ExperimentErliyani Sartono1*, Ida M. Lisse2, Elisabeth M. Terveer1, Paula J. M. van de Sande1, Hilton Whittle3, Ane B.

Fisker4, Adam Roth5, Peter Aaby6, Maria Yazdanbakhsh1, Christine S. Benn4

1 Department of Parasitology, Leiden University Medical Center, Leiden, the Netherlands, 2 Department of Pathology, Herlev University Hospital, Herlev, Denmark, 3 MRC

Laboratories, Fajara, Gambia, 4 Bandim Health Project, Statens Serum Institut, Copenhagen, Denmark, 5 Department of Medical Microbiology, Lund University, Lund,

Sweden, 6 Bandim Health Project, Indepth Network, Bissau, Guinea-Bissau

Abstract

Background: Oral polio vaccine (OPV) is recommended to be given at birth together with BCG vaccine. While we wereconducting two trials including low-birth-weight (LBW) and normal-birth-weight (NBW) infants in Guinea-Bissau, OPV wasnot available during some periods and therefore some infants did not receive OPV at birth, but only BCG. We investigatedthe effect of OPV given simultaneously with BCG at birth on the immune response to BCG vaccine.

Methods and Findings: We compared the in vitro and the in vivo response to PPD in the infants who received OPV and BCGwith that of infants who received BCG only. At age 6 weeks, the in vitro cytokine response to purified protein derivate (PPD)of M. Tuberculosis was reduced in LBW and NBW infants who had received OPV with BCG. In a pooled analysis receiving OPVwith BCG at birth was associated with significantly lower IL-13 (p = 0.041) and IFN-c (p = 0.004) and a tendency for lower IL-10 (p = 0.054) in response to PPD. Furthermore, OPV was associated with reduced in vivo response to PPD at age 2 months,the prevalence ratio (PR) of having a PPD reaction being 0.75 (0.58–0.98), p = 0.033, and with a tendency for reducedlikelihood of having a BCG scar (0.95 (0.91–1.00), p = 0.057)). Among children with a scar, OPV was associated with reducedscar size, the regression coefficient being 20.24 (20.43—0.05), p = 0.012.

Conclusions: This study is the first to address the consequences for the immune response to BCG of simultaneousadministration with OPV. Worryingly, the results indicate that the common practice in low-income countries ofadministering OPV together with BCG at birth may down-regulate the response to BCG vaccine.

Citation: Sartono E, Lisse IM, Terveer EM, van de Sande PJM, Whittle H, et al. (2010) Oral Polio Vaccine Influences the Immune Response to BCG Vaccination. ANatural Experiment. PLoS ONE 5(5): e10328. doi:10.1371/journal.pone.0010328

Editor: Lisa F. P. Ng, Singapore Immunology Network, Singapore

Received December 18, 2009; Accepted April 2, 2010; Published May 21, 2010

Copyright: � 2010 Sartono et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The study was supported by the European Commission, INCO-programme (contract no. ICA4-CT-2002-10053). The funders had no role in study design,data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

WHO recommends BCG vaccine and oral polio vaccine (OPV)

at birth in low-income countries. BCG vaccine is the world’s most

widely used vaccine and although its efficacy is variable, it is still

considered the best way to protect against childhood tuberculosis

[1,2]. BCG vaccination induces potent Th1 responses to mycobac-

terial antigen in newborns [3]. Studies in Gambia have shown that

BCG vaccination affects responses to unrelated vaccines by

enhancing the production of Th1 and Th2 type cytokines [3,4].

BCG vaccination in early life also increased the antibody response

to OPV [4]. The effect of providing OPV simultaneously with BCG

on the immune response to BCG has never been studied.

We recently conducted two trials among newborns in Guinea-

Bissau. According to local practice, low-birth-weight (LBW,

,2500 g) infants only receive OPV at birth; BCG vaccine is

usually postponed until the child reaches 6 weeks of age. In trial 1,

LBW infants were randomized to receive an early BCG vaccine or

the usual delayed BCG vaccine. In trial 2, normal birth-weight

(NBW, $2500 g) children were randomized to vitamin A

supplementation or placebo together with BCG vaccine [5]. The

children were followed for mortality. Subgroups of the children

were also followed for the in vivo and in vitro immune response to

BCG [6]. OPV is recommended to be given at birth but for

certain periods in 2004, during the conduct of the two trials, OPV

was not available in Guinea-Bissau and hence some trial

participants did not receive OPV at birth. This ‘natural

experiment’ allowed us to compare in an observational, but rather

unbiased manner, the effect of OPV provided with BCG at birth.

Intriguingly, we observed that not receiving OPV at birth was

associated with significantly decreased male mortality during the

first year of life [7]. In the present study we investigated the effect

of OPV given simultaneously with BCG at birth on the immune

response to BCG vaccine.

Materials and Methods

Ethics StatementThis study was conducted according to the principles expressed

in the Declaration of Helsinki. The study was approved by the

PLoS ONE | www.plosone.org 1 May 2010 | Volume 5 | Issue 5 | e10328

research committee of the Ministry of Health in Guinea-Bissau

and for European Centers by the Danish Central Ethical

Committee. All mothers provided written informed consent for

the collection of samples of their children.

SettingThe two large randomized trials were conducted by the Bandim

Health Project (BHP) in Bissau, Guinea-Bissau. The BHP covers a

population of around 100,000 in 6 districts in the urban area by a

demographic surveillance system. All houses are visited every month

to register new pregnancies and births. Once a newborn is

identified, the child is followed with a home visit every third month.

There is a national hospital (NH) with a maternity ward in the

capital and three health centers in the study area, one of them with a

maternity ward. Around 60–70% of the mothers from the study

area deliver at the NH or at the health center maternity ward. Since

2002 all LBW infants born at the NH have been followed for one

year, also infants outside the study area. In trial 1, all LBW infants

born at the NH or in the study area were invited to participate in a

trial studying the effect of receiving early BCG vaccination on

mortality and cytokine levels in LBW infants. In trial 2, NBW

infants from the study area were invited to participate in a trial

studying the effect of vitamin A supplementation at birth on

mortality and cytokine levels. These two EU funded trials were

carried out within a collaborative network of European and Guinea-

Bissau institutions. The trials were registered at www.clinicaltrials.

gov, registration number, NCT00146302 and NCT00168597.

Enrolment into the trialsNewborns were enrolled throughout 2004 (Figure 1). Mothers

were informed of the purpose of the study. If a mother of a LBW

infant accepted to participate, she drew a randomization number

indicating whether the children would be BCG vaccinated early or

not. If a mother of a NBW infant accepted to participate, she drew

a randomization number indicating whether the children would

receive vitamin A supplementation (50,000 IU) or placebo. At the

time of enrolment, children were examined clinically. The infants

were weighed and length, head-circumference and mid-upper-

arm-circumference (MUAC) were measured. Newborns that were

overtly ill were not enrolled but referred to treatment.

LBW infants who were randomized to not receiving early BCG

vaccination were not included in the present study since we aimed

to look at the effect of OPV on the immune response to BCG

given at the same time. Hence, the LBW population only includes

children who were randomized to BCG at birth. NBW children

who had received vitamin A supplementation at birth were not

included in the present study as this could have affected their

response to OPV and BCG. Hence, the NBW population only

includes children who were randomized to placebo at birth.

VaccinesBCG (0.05 ml; Statens Serum Institut, Copenhagen, Denmark)

was provided by the BHP and given intradermally in the upper left

deltoid region. OPV was supplied through the national immuni-

zation programme and given orally. Three types of OPV were

used in Guinea-Bissau, produced by Novartis, Sanofi Pasteur, and

GlaxoSmithKline. However, OPV was missing during periods of

2004. As part of our trial routines, we noted at the inclusion form

whether the children received OPV or not. Based on the

distribution of children who had not received OPV, OPV was

missing at one or more inclusion sites 1) from the beginning of

February 2004 to the beginning of June 2004, 2) again in the end

of June 2004, and 3) from the end of October 2004 to November

2004 (Figure 1).

Figure 1. An overview of the two trials, the periods with missing OPV, and the periods with ongoing subgroup studies in Guinea-Bissau 2004.doi:10.1371/journal.pone.0010328.g001

OPV Reduces BCG Responses


Follow-up for in vitro PPD responseWe aimed to enroll 200 children from trial 1 and 400 children

from trial 2 in a subgroup study of cytokine production. The

children were enrolled between April and November 2004

(Figure 1). We attempted to visit the children at 6 weeks of age. A

blood sample was collected by finger prick and analyzed for in vitro

cytokine responses. Whole blood cultures were done as previously

described [6,8]. Briefly, heparinized finger prick blood was diluted

in RPMI 1640 medium (Invitrogen, Breda, The Netherlands) to a

final concentration of 1 in 10 and culture in a total volume of 200 ml

in 96-well U plate (Nunc, Roskilde, Denmark) with the presence of

purified protein derivative of Mycobacterium tuberculosis (PPD, 10 mg/

ml; Statens Serum Institut, Denmark, Copenhagen). Supernatants

were collected on day 3 and stored at 280uC.

Measurement of cytokine levelsIL-5, IL-10, IL-13, IFN-c and TNF-a from day 3 supernatants

were analyzed simultaneously using commercial Luminex

cytokine kit and buffer reagent kit (BioSource, Camarillo, CA,

USA) and run on a Luminex-100 cytometer (Luminex Corpo-

ration, Austin, TX, USA), equipped with StarStation software

(Applied Cytometry Systems, Dinnington, UK). The assay was

performed with slight modification from the manufacturer’s

recommendation. Briefly, assays were done in a 96-well U plate

at room temperature. Mixes of beads were incubated for 2 hours

with a standard, samples, or blank in a final volume of 50 ml for

2 hours under continuous shaking. Beads were washed twice and

incubated with cocktail of biotinylated antibodies (25 ml/well) for

1 hour. After removal of excess biotinylated antibodies, Strepta-

vidin-PE was added for 30 minutes. The plate was then washed

and analyzed. The lower detection limit of the assays was 3 pg/

ml, 5 pg/ml, 10 pg/ml, 5 pg/ml and 10 pg/ml for IL-5, IL-10,

IL-13, IFN-c and TNF-a, respectively. Samples with concentra-

tions below the detection limit were given the value of this

threshold.

Follow-up for BCG scar and in vivo PPD responseAll LBW children were visited at home and examined for BCG

scar and in vivo PPD response at 2 and 6 months of age. In four of

six study area districts NBW children were examined for BCG

scar and in vivo PPD response when they reached 2 and 6 months

of age. At the home visits a trained nurse documented health

status and vaccination status. She measured the size of the scar

following BCG vaccination. Subsequently, 0.1 ml of Tuberculin

PPD RT23 (Statens Serum Institut, Copenhagen, Denmark) was

injected intradermally on the ventral side of the forearm. Between

48 and 72 hours later, the child was visited again, and the

induration was measured by a trained field worker using track-

ball technique [9]. Size of scar and induration was defined as the

average of the height and the width measured to nearest

millimeter with a transparent ruler. Children with a measurable

scar were categorized as ‘‘scar-positive’’. Children with a mean

diameter of the induration $2 mm were categorized as ‘‘PPD-

positive’’. Children who had taken chloroquine within seven days

prior to the visit were excluded from the PPD analysis, since

chloroquine treatment is known to suppress PPD reaction [10].

These children were retained in the analysis of scar reaction since

we considered it unlikely that current chloroquine treatment

would have affected the scar formation. Children who had

received BCG vaccination less than 30 days prior to the visit or

with known exposure to tuberculosis in the household within the

previous 6 months (information only available for NBW

population) were excluded. Children with PPD reactions

.10 mm were referred to a medical doctor to be further

examined for tuberculosis (TB).

Statistical analysisAll statistical analysis was performed in Stata 10.0.

In vitro PPD response. Cytokine levels were not normally

distributed. The crude cytokine levels were first compared in a

univariate analysis using non-parametric Mann-Whitney U tests,

and the results were provided in the figures of the distribution of

cytokine levels in the different groups (Figures 2 and 3).

Subsequently, the frequency of low and high cytokine

producers in BCG and BCG+OPV recipients was compared in

Poisson regression model with robust variance estimates [11]

using the median cytokine level in the total population as cut off

for low and high responder. Thus, the relative measure is a

prevalence ratio (PR). The study population was divided into 4

groups according to the birth weight and whether OPV was given

together with BCG at birth (Table 1). As the periods with lack of

OPV fell primarily in the dry season (from December to May), all

children who had received BCG+OPV had been vaccinated in

the rainy season (June to November). The two LBW groups were

similar with respect to age and sex. However, MUAC at

enrolment was larger in the BCG+OPV group compared with

the BCG group, and they had received more OPV and more

diphtheria-tetanus-pertussis (DTP) vaccines (recommended at age

6, 10 and 14 weeks) between enrolment and blood sampling

(Table 1). The two NBW groups were similar with respect to age

and MUAC at enrolment (Table 1). However, they were

significantly different with regard to sex, the NBW BCG+OPV

group having fewer boys. The two groups also differed

significantly with regard to vaccinations received between

enrolment and blood sampling (more OPV and less DTP in

the BCG+OPV group). All further analyses were adjusted for age,

sex, month of enrolment, MUAC at enrolment, and vaccinations

between enrolment and blood sampling.

BCG scar and in vivo PPD response. PPD response (yes/

no) and scar response (yes/no) were analyzed as prevalence data

using a Poisson regression model with robust variance estimates

providing prevalence ratios (PR) [11]. Children with a PPD

response above 15 mm were excluded from the analyses as they

had potentially been exposed to TB. There were no such children

among LBW children. Among NBW children one boy

(BCG+OPV) had a PPD response above 15 mm at age 2

months and was excluded from further analyses. At age 6

months, one boy (BCG+OPV) and one girl (BCG), had a PPD

response above 15 mm and were excluded from the 6 month-

analysis. As in the cytokine analysis, there was a clear overweight

of children who had received BCG+OPV in the rainy season,

though there was more variation because the follow-up for PPD/

scar had been conducted throughout 2004 (Figure 1). Among

LBW infants, the BCG+OPV recipients only differed from the

BCG only recipients with regard to season (Table 2). Among NBW

infants, the two groups also differed with regard to MUAC at

enrolment, and number of OPV received between enrolment and

the PPD/Scar assessment (Table 2). We adjusted the analyses for

sex, season (the larger variation in this analysis compared with the

cytokine analysis allowed us to control for season rather than

month), MUAC, and subsequent vaccinations. As in previous

studies of PPD responses, the reading of the PPD reaction differed

significantly by the assistant doing the reading and the analyses of

PPD responses were therefore furthermore adjusted for assistant.

Among PPD responders and/or scar reactors, we analyzed the size

of the reaction in linear regression models controlling for the same

potential confounders.



Results

In vitro PPD responseThis analysis included 61 LBW infants who had been

randomized to early BCG; 33 who had not received OPV at

birth together with BCG and 28 who had. Furthermore, 248

NBW infants who had been randomized to placebo; 69 who had

not received OPV and 179 who had were included.

Influence of OPV on cytokine responses to PPD in LBWinfants

When OPV was given together with BCG, there was a

significant down regulation of IL-5, IL-13, and IFN-c in response

to PPD as compared to the group of infants who had received

BCG only (Figure 2)(Table 3). IL-10 to PPD was higher in LBW

infants who had received BCG together with OPV in the crude

Figure 2. PPD stimulated IFN-c (A), IL-5 (B), IL-13 (C), TNF-a (D) and IL-10 (E) production in low birth weight BCG (LBW BCG) andBCG+OPV vaccinated (LBW BCG+OPV) infants. One dot represents one individual. Horizontal lines represent medians.doi:10.1371/journal.pone.0010328.g002

Figure 3. PPD stimulated IFN-c (A), IL-5 (B), IL-13 (C), TNF-a (D) and IL-10 (E) production in normal birth weight BCG (NBW BCG) andBCG+OPV vaccinated (NBW BCG+OPV) infants. One dot represents one individual. Horizontal lines represent medians.doi:10.1371/journal.pone.0010328.g003



analysis, but the difference disappeared in the adjusted analysis

(Table 3). There were no significant differences in TNF-a to PPD

between the two groups (Figure 2)(Table 3).

Influence of OPV on cytokine responses to PPD in NBWinfants

In the unadjusted analysis IFN-c responses to PPD appeared

depressed and IL-10 responses increased when OPV was given

with BCG (Figure 3). In the adjusted analysis, the IFN-c levels to

PPD were significantly lower in BCG+OPV compared with BCG

only vaccinated infants and there was a similar tendency for IL-13

(Table 3).

Influence of OPV on cytokine responses to PPD in allinfants

In a pooled analysis of LBW and NBW infants, receiving OPV

with BCG at birth was associated with significantly lower IL-13

and IFN-c in responses to PPD; the adjusted PRs (furthermore

adjusted for being LBW) were 0.64 (0.41–0.98)(p = 0.041), and

0.51 (0.33–0.80)(p = 0.004), respectively. The IL-10 response to

PPD tended to be lower as well (0.61 (0.36–1.01), p = 0.054).

BCG scar and in vivo PPD responseAmong LBW infants enrolled in 2004, 250 BCG recipients were

visited; 104 had received BCG+OPV, 146 BCG only. We

obtained a scar reading in 231 children at age 2 months and

200 children at age 6 months and a valid PPD assessment of 202 at

age 2 months, and 170 at age 6 months. Among NBW infants

enrolled in 2004, 540 placebo recipients had their BCG scar and

PPD response assessed once or more; 287 had received

BCG+OPV; 253 BCG only. We obtained a scar reading in 438

children at age 2 months and 428 children at age 6 months and a

valid PPD assessment of 379 at age 2 months, and 327 at age 6

months.

Influence of OPV on PPD/Scar responses in LBW

infants. At age 2 months, receiving OPV with BCG at birth was

associated with a borderline significantly lower likelihood of being

PPD positive (Table 4). There was no association with size of the

reaction among the few children who had a positive reaction.

Almost all children were scar positive, and there was no association

between OPV at birth and being scar positive (Table 5), or the size

of the scar among scar positives (data not shown).

At age 6 months, the lower likelihood of being PPD positive

(Table 4) was no longer significant for the OPV group and there

was no association with size of the reaction among the few children

who had a positive reaction. Almost all children were scar positive,

and there was no association between OPV at birth and being scar

positive (Table 5), or the size of the scar among scar positives (data

not shown).

Influence of OPV on PPD/Scar responses in NBW

infants. At age 2 months, receiving OPV with BCG at birth

was not associated with the likelihood of being PPD positive

(Table 4), nor was it associated with the size of the response among

children with a positive response (data not shown). Adjusted for

potential confounders, children who had received OPV at birth

had a lower likelihood of developing a BCG scar (Table 5).

Receiving OPV at birth was negatively associated with the size of

the scar among those who had a scar (regression coefficient 20.36

(20.57—0.15); p = 0.001).

Table 2. Description of the scar and PPD study population.

Group N (boys/girls)Vaccinated inrainy season

Median MUAC atvaccination (10–90centiles)

Received OPVbetweenvaccination andPPD assessment

Received DTPbetweenvaccination andPPD assessment

Median age atsampling (10–90centiles)

Low-birth-weight infants

BCG only 146 (57/89) 49 (34%) 82 mm (70–88) 61 (42%) 103 (71%) 65 days (57–79)

BCG+OPV 104 (43/61) 66 (63%) 82 mm (70–90) 47 (45%) 74 (71%) 64 days (59–75)

Normal-birth-weight infants

BCG only 253 (143/110) 74 (29%) 96 mm (88–110) 135 (53%) 230 (91%) 63 days (54–82)

BCG+OPV 287 (146/141) 228 (79%) 100 mm (88–112) 184 (64%) 257 (90%) 64 days (55–78)

doi:10.1371/journal.pone.0010328.t002

Table 1. Description of the cytokine study population.

Group N (boys/girls)Vaccinated inrainy season

Median MUACat BCG vaccination(10–90 centiles)

Received OPVbetweenvaccination andblood sample

ReceivedDTP betweenvaccinationand blood sample

Median ageat sampling(10–90 centiles)


BCG only 33 (11/22) 15 (45%) 82 mm (70–88) 3 (9%) 2 (6%) 44 days (38–49)

BCG+OPV 28 (8/20) 28 (100%) 82 mm (70–94) 17 (61%) 7 (25%) 48 days (34–56)


BCG only 69 (44/25) 12 (17%) 100 mm (88–112) 24 (35%) 20 (29%) 43 days (37–59)

BCG+OPV 179 (77/102) 179 (100%) 100 mm (88–108) 122 (68%) 26 (15%) 42 days (36–54)

doi:10.1371/journal.pone.0010328.t001



At age 6 months, receiving OPV with BCG at birth was not

associated with the likelihood of being PPD positive (Table 4) or

the size of the PPD reaction (data not shown). Almost all children

had a scar at age 6 months and receiving OPV at birth was not

associated with the likelihood of being scar positive (Table 5) or the

size of the scar (data not shown).

Influence of OPV on PPD/Scar responses in all

infants. In a pooled analysis of LBW and NBW infants,

receiving OPV with BCG at birth was associated with

significantly lower likelihood of being PPD positive at age 2

months, the PR being 0.75 (0.58–0.98), p = 0.033. OPV with BCG

was not associated with size of the PPD reaction (data not shown).

Table 3. Comparison of the frequency of cytokine responders in children vaccinated with BCG and with BCG+OPV.

Response to PPD Proportion of high responders (%)Prevalence Ratio (95% CI)BCG+OPV versus BCG only

Adjusted Prevalence Ratio (95% CI)BCG+OPV versus BCG only

BCG+OPV BCG only


(N = 28) (N = 33)

IL-5 9 (32%) 21 (64%) 0.51 (0.28–0.92) 0.56 (0.28–1.12)

IL-10 17 (61%) 11 (33%) 1.82 (1.03–3.23) 0.46 (0.14–1.49)

IL-13 13 (46%) 23 (70%) 0.67 (0.42–1.06) 0.51 (0.22–1.16)

IFN-c 12 (43%) 24 (73%) 0.59 (0.36–0.95) 0.40 (0.15–1.09)

TNF-a 13 (46%) 20 (61%) 0.77 (0.47–1.25) 2.25 (0.74–6.85)

Normal birth-weight infants

(N = 179) (N = 69)

IL-5 83 (46%) 46 (58%) 0.80 (0.62–1.03) 0.73 (0.42–1.26)

IL-10 96 (54%) 31 (45%) 1.19 (0.89–1.60) 0.63 (0.32–1.21)

IL-13 82 (46%) 36 (52%) 0.88 (0.67–1.16) 0.66 (0.38–1.15)

IFN-c 78 (44%) 41 (59%) 0.73 (0.57–0.95) 0.54 (0.30–0.97)

TNF-a 87 (49%) 34 (49%) 0.99 (0.74–1.31) 0.88 (0.40–1.95)

Adjusted for age, sex, MUAC and month of enrolment, subsequent vaccinations.doi:10.1371/journal.pone.0010328.t003

Table 4. The effect of receiving OPV with BCG at birth on the likelihood of having an in vivo response to PPD at age 2 months and6 months.

ALL CHILDRENCRUDE PR (95% CI)BCG+OPV VERSUS BCG ONLY

ADJUSTED PR (95% CI)BCG+OPV VERSUS BCG ONLY

Positive/all (%)


2 months

BCG+OPV 7/87 (8%) 0.49 (0.21–1.11) 0.43 (0.18–1.02)**

BCG only 19/115 (17%)

6 months

BCG+OPV 17/71 (24%) 0.85 (0.50–1.43) 0.65 (0.39–1.13)

BCG only 28/99 (28%)


2 months

BCG+OPV 70/208 (34%) 0.81 (0.62–1.05) 0.83 (0.62–1.09)

BCG only 71/170 (42%)

6 months

BCG+OPV 56/167 (34%) 1.01 (0.74–1.38) 1.09 (0.75–1.59)

BCG only 52/157 (33%)

Adjusted for sex, MUAC and season of enrolment, subsequent vaccinations and assistant.**P = 0.056.doi:10.1371/journal.pone.0010328.t004



OPV with BCG was also tended to be associated with a reduced

likelihood of being scar positive (0.95 (0.91–1.00), p = 0.057).

Among scar positive children, OPV was associated with

significantly reduced scar size, the regression coefficient being

20.24 (20.43—0.05), p = 0.012 (Figure 4). No significant

differences were seen at age 6 months.

Discussion

This study is the first to demonstrate that mucosal administra-

tion of OPV simultaneously with intradermal BCG vaccination

may have profound immunological consequences. By comparing

immune responses of infants who had received BCG at birth either

with or without OPV, we observed that OPV had a significant

suppressing effect on Th1 and Th2 responses to PPD. These in vitro

findings were paralleled by findings of significantly reduced in vivo

responses to PPD, a tendency for less BCG scars, and significantly

reduced BCG scar size among those who developed a scar.

Strengths and weaknessesThough OPV is widely used, little is known regarding the

cellular immune responses following vaccination, in particular its

non-specific immune responses to un-related stimuli. The effect of

OPV could be studied in the present study due to a temporary

shortage of the vaccine in Guinea-Bissau during several periods of

2004. The shortage occurred in a period when two trials,

examining among other things the immune responses of

newborns, were being conducted. It was therefore possible to

study in a relatively unbiased manner the effect of OPV on the in

vitro response to PPD, as well as the effect on two markers of BCG

immunity: PPD responses and BCG scarification.

Strengths of our study include the design; it was mainly logistic

factors which determined whether the children would receive

OPV or not. Despite this, there were some clear differences

between the children who did or did not receive OPV at birth for

instance with regard to season of vaccination, anthropometrics at

inclusion, and other vaccines received. We conducted our analyses

with and without adjustment for these co-variables, and indeed

noted that the crude estimated changed considerably when we

included the other variables as evidence of confounding. We

cannot exclude that there is considerable residual confounding due

to for instance the burden of maternal infectious diseases, other

vaccines received or nutritional status. Especially, residual

confounding by season could be a problem. A recent study in

Malawi found that in vitro IFN-c response to PPD varied strongly

by season, being lowest in the warm and rainy season as compared

with the cool and dry season and the hot and dry season [12]. In

Guinea-Bissau there are two distinct seasons. Our periods without

OPV coincided with the dry season. Hence, we have largely

compared children who received OPV in the rainy season with

children who received no OPV in the dry season. If the IFN-cresponse to PPD was depressed for other reasons in the rainy

Table 5. The effect of receiving OPV with BCG at birth on the likelihood of being scar positive at age 2 and 6 months.

ALL CHILDRENPR (95% CI) BCG+OPVVERSUS BCG ONLY

ADJUSTED PR (95% CI)BCG+OPV VERSUS BCG ONLY

Positive/all (%)


2 months

BCG+OPV 90/97 (93%) 1.00 (0.93–1.08) 1.00 (0.92–1.08)

BCG only 124/134 (93%)

6 months

BCG+OPV 74/78 (95%) 0.98 (0.92–1.04) 0.98 (0.92–1.05)

BCG only 118/122 (97%)


2 months

BCG+OPV 203/227 (89%) 0.95 (0.90–1.00) 0.93 (0.87–0.99)**

BCG only 198/210 (94%)

6 months

BCG+OPV 217/227 (96%) 0.98 (0.95–1.02) 0.99 (0.95–1.04)

BCG only 193/198 (97%)

Adjusted for sex, MUAC and season of enrolment, and subsequent vaccinations.**P = 0.015.doi:10.1371/journal.pone.0010328.t005

Figure 4. Comparison of BCG scar size at age 2 monthsbetween infants who got or did not get OPV with BCG at birth.doi:10.1371/journal.pone.0010328.g004



season it could be at least part of the reason for the observed lower

IFN-c response to PPD response in OPV recipients. However, we

found the same results when we limited our analysis to the rainy

season, though the confidence intervals of course became wider

(data not shown). Furthermore, we found depressed in vivo

responses to PPD as well. The PPD data set was larger than the

cytokine data set, and had more seasonal variation and thereby

more power to control adequately for season. The validity of our

findings is supported by the fact that the findings from the LBW

and NBW cohorts consistently pointed in the same direction, and

the pooled analyses provided significant results. Nonetheless, our

findings should be interpreted with caution.

Previously, it has been shown that BCG vaccination given at

birth in either NBW or LBW infants enhanced immune response

to PPD, characterized by elevated IL-5, IL-13 and IFN-c levels (4,

and unpublished data). In the present study, the administration of

OPV at the time of BCG priming at birth, reduced Th1 and Th2

cytokine responses to mycobacterial antigens in LBW infants,

suggesting that OPV down regulated cellular immune responses to

BCG vaccine when the vaccines were administrated at the same

time.

One possible mechanism whereby OPV co administered with

BCG leads to a reduced cellular response to PPD may be

competition of OPV and BCG activated T cells for resources and

space [13,14]. A second possible mechanism is that polio virus

may have specific immune modulatory molecules that down

regulate immune responses to antigens to which immune

responses are being mounted simultaneously [15].

The fact that the downregulation of cytokine production to PPD

as a result of OPV vaccination was more profound in LBW

compared to NBW infants might be due to functional immaturity

of LBW infants. A recent study by Klein et al. showed that after

inactivated polio vaccine, preterm infants had comparable

frequencies of poliovirus specific CD4+CD45RO+CD69+IFN-cmemory T cells but diminished lymphoproliferation compared to

term infants [16]. Furthermore, it is also possible the differences

between LBW and NBW infants were caused by differences in the

genetic make up. A study by Anuradha et al. reported that IFN-clow producer genotype +874T/A were over represented in BCG

non-responding children [17].

Implications for BCG immunityThere is considerable evidence, both in humans and animals

that IFN-c production is necessary for protective immunity against

mycobacterial disease [18–21] and successful chemotherapy of TB

is associated with augmented IFN-c production [22–24]. Further-

more, it has also been reported that individuals with genetic

abnormalities of the IFN-c receptor are highly susceptible to

intracellular infections [25–27]. Thus, immunity to tuberculosis

might be impaired by the down regulation of this Th1 response in

LBW children receiving OPV at the same time as BCG at birth.

The finding is supported by the fact that we found reduced PPD

responses and reduced BCG scarification and scar size at age 2

months although these are debatable markers for TB immunity.

Interestingly, the differences were no longer significant at age 6

months. It could be speculated that the PPD skin test at 2 months

of age boosted the immune response to BCG and this obliterated

the differences at 6 months. It has previously been reported that

PPD skin test boosts the subsequent skin test reactivity [28]. The

clinical implications of this early depression in immune responses

to BCG need to be addressed in well-designed randomized trials

with sufficient follow-up.

It is worth noticing that the protection induced by BCG against

tuberculosis has been found to be much higher in high-income

countries than in low-income countries. High-income countries

mainly administer inactivated polio vaccine (IPV) at several

months of age. Though many of the BCG-efficacy studies were

done many years ago, before the introduction of OPV at birth, it

may be speculated that the addition of OPV to BCG at birth has

further compromised the protection induced by BCG in low-

income countries.

ConclusionsIn summary, we are aware that the study population is small,

and though we were able to control for a number of confounders

there may be residual confounding. However, the potential effect

of OPV on the immune response to BCG vaccine has not been

studied before, and it may seem biologically plausible that the two

vaccines interact with each other. The potentially negative effect of

OPV on the immune response to BCG vaccine should be tested in

a randomized trial.

Acknowledgments

We are indebted to all study participants at the Bandim Health Project

(BHP) in Bissau, Guinea-Bissau.

Author Contributions

Conceived and designed the experiments: HCW PA MY. Performed the

experiments: ES IML EMT PJMvdS ABF AR CSB. Analyzed the data: ES

IML ABF AR CSB. Wrote the paper: ES CSB.

References

1. Colditz GA, Brewer TF, Berkey CS, Wilson ME, Burdick E, et al. (1994)

Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the

published literature. JAMA 271: 698–702.

2. Fine PE (1995) Variation in protection by BCG: implications of and for

heterologous immunity. Lancet 346: 1339–45.

3. Marchant A, Goetghebuer T, Ota MO, Wolfe I, Ceesay SJ, et al. (1999)

Newborns develop a Th1-type immune response to Mycobacterium bovis

bacillus Calmette-Guerin vaccination. J Immunol 163: 2249–55.

4. Ota MO, Vekemans J, Schlegel-Haueter SE, Fielding K, Sanneh M, et al. (2002)

Influence of Mycobacterium bovis bacillus Calmette-Guerin on antibody and

cytokine responses to human neonatal vaccination. J Immunol 168: 919–25.

5. Benn CS, Diness BR, Roth A, Nante E, Fisker AB, et al. (2008) Effect of 50,000

IU vitamin A given with BCG vaccine on mortality in infants in Guinea-Bissau:

randomised placebo controlled trial. BMJ 336: 1416–20.

6. Diness BR, Fisker AB, Roth A, Yazdanbakhsh M, Sartono E, et al. (2007) Effect

of high-dose vitamin A supplementation on the immune response to Bacille

Calmette-Guerin vaccine. Am J Clin Nutr 86: 1152–9.

7. Benn CS, Fisker AB, Rodrigues A, Ravn H, Sartono E, et al. (2008) Sex-

differential effect on infant mortality of oral polio vaccine administered with

BCG at birth in Guinea-Bissau. A natural experiment. PLoS ONE 3(12): e4056.

8. Eriksson M, Sartono E, Martins CL, Bale C, Garly ML, et al. (2007) A

comparison of ex vivo cytokine production in venous and capillary blood. Clin

Exp Immunol 150: 469–76.

9. Bouros D, Zeros G, Panaretos C, Vassilatos C, Siafakas N (1999) Palpation vs

pen method for the measurement of skin tuberculin reaction (Mantoux test).

Chest 99: 416–9.

10. Garly ML, Bale C, Martins CL, Balde MA, Hedegaard KL, et al. (2001) BCG

vaccination among West African infants is associated with less anergy to

tuberculin and diphtheria-tetanus antigens. Vaccine 20: 468–74.

11. Barros AJ, Hirakata VN (2003) Alternatives for logistic regression in cross-

sectional studies: an empirical comparison of models that directly estimate the

prevalence ratio. BMC Med Res Methodol 3: 21.

12. Lalor MK, Ben-Smith A, Gorak-Stolinska P, Weir RE, Floyd S, et al. (2009)

Population differences in immune responses to Bacille Calmette-Guerin

vaccination in infancy. J Infect Dis 199: 795–800.

13. Gunther J, Haas W, Von Boehmer H (1982) Suppression of T cell responses

through competition for T cell growth factor (interleukin 2). Eur J Immunol 12:

247–9.

14. Stockinger B, Kassiotis G, Bourgeois C (2004) CD4 T-cell memory. Semin

Immunol 16: 295–303.



15. Vekemans J, Ota MO, Wang EC, Kidd M, Borysiewicz LK, et al. (2002) T cell

responses to vaccines in infants: defective IFNgamma production after oral polio

vaccination. Clin Exp Immunol 127: 495–8.

16. Klein NP, Gans HA, Sung P, Yasukawa LL, Jhonson J, et al. (2010) Preterm

infants’ T cell responses to inactivated poliovirus vaccine. J Infect Dis 201:

214–22.

17. Anuradha B, Rakh SS, Ishaq M, Murthy KJR, Valluri VL (2008) Interferon-clow producer genotype +874 overrepresented in Bacillus Calmette-Guirin

nonresponding children. Pediatr Infect Dis J 27: 325–9.

18. Cooper AM, Dalton DK, Stewart TA, Griffin JP, Russell DG, et al. (1993)

Disseminated tuberculosis in interferon gamma gene-disrupted mice. J Exp Med

178: 2243–7.

19. Flynn JL, Chan J, Triebold KJ, Dalton DK, Stewart TA, et al. (1993) An

essential role for interferon gamma in resistance to Mycobacterium tuberculosis

infection. J Exp Med 178: 2249–54.

20. Jouanguy E, Altare F, Lamhamedi S, Revy P, Emile JF, et al. (1996) Interferon-

gamma-receptor deficiency in an infant with fatal bacille Calmette-Guerin

infection. N Engl J Med 335: 1956–61.

21. Newport MJ, Huxley CM, Huston S, Hawrylowicz CM, Oostra BA, et al. (1996)

A mutation in the interferon-gamma-receptor gene and susceptibility to

mycobacterial infection. N Engl J Med 335: 1941–9.

22. Al-Attiyah R, Mustafa AS, Abal AT, Madi NM, Andersen P (2003) Restoration

of mycobacterial antigen-induced proliferation and interferon-gamma responses

in peripheral blood mononuclear cells of tuberculosis patients upon effective

chemotherapy. FEMS Immunol Med Microbiol 38: 249–56.23. Hirsch CS, Toossi Z, Othieno C, Johnson JL, Schwander SK, et al. (1999)

Depressed T-cell interferon-gamma responses in pulmonary tuberculosis:

analysis of underlying mechanisms and modulation with therapy. J Infect Dis180: 2069–73.

24. Marchant A, Amedei A, Azzurri A, Vekemans J, Benagiano M, et al. (2001)Polarization of PPD-specific T-cell response of patients with tuberculosis from

Th0 to Th1 profile after successful antimycobacterial therapy or in vitro

conditioning with interferon-alpha or interleukin-12. Am J Respir Cell Mol Biol24: 187–94.

25. Allende LM, Lopez-Goyanes A, Paz-Artal E, Corell A, Garcıa-Perez MA, et al.(2001) A point mutation in a domain of gamma interferon receptor 1 provokes

severe immunodeficiency. Clin Diagn Lab Immunol 8: 133–7.26. Cunningham JA, Kellner JD, Bridge PJ, Trevenen CL, Mcleod DR, et al. (2000)

Disseminated bacille Calmette-Guerin infection in an infant with a novel

deletion in the interferon-gamma receptor gene. Int J Tuberc Lung Dis 4:791–4.

27. Dorman SE, Holland SM (1998) Mutation in the signal-transducing chain of theinterferon-gamma receptor and susceptibility to mycobacterial infection. J Clin

Invest 101: 2364–9.

28. Dogan E, Erkoc R, Sayarlioglu H, Uzun K (2005) Tuberculin skin test resultsand the booster phenomenon in two-step tuberculin skin testing in hemodialysis

patients. Ren Fail 27: 425–8.



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